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. 2015 Sep 22;10(9):e0138789. doi: 10.1371/journal.pone.0138789

Fig 5. CBLC ubiquitin ligase activity is required for Golgi but not SRC levels regulation.

Fig 5

(A) Requirement of CBLC RING domain. HeLa wild type (WT) or stably expressing siRNA-resistant-CBLC (CBLC-GFP) or siRNA-resistant-CBLC RING mutant (C351A-GFP) cells were transfected with deconvoluted CBLC-7 siRNA. Golgi stained with Giantin. Top right: Schematic diagram, exogenous CBLC contains three-point mutations at CBLC-7 siRNA-binding site. Bottom right: Fragmentation Index measured in quadruplicates on at least 400 cells per condition. Error bars show SD statistical significance (p) measured by unpaired Student’s t-test. (**) represents p<0.01 relative to non-target siRNA transfected cells. Scale bar = 10μm. (B) SRC levels analysis by western blot upon CBLC silencing or overexpressing. (C) CBLC depletion does not activate GALNTs relocation. pY20 immunostaining marks SRC-activated cells, Golgi labelled with Giantin, and GALNTs activity revealed by HPL staining. HeLa cells transfected with non-target siRNA, CBLC siRNA, or constitutively active SRC E378G plasmid, or stimulated with PDGF. Scale bar = 10 μm.